Human growth hormone (“hGH”) also known as somatotropin consists of 191 amino acids with a molecular mass of 22 kDa, with two disulfide bridges between the chains, which is synthesized and secreted by cells called somatotrophs in the anterior pituitary. Growth hormone is produced in the anterior lobe or in the glandular portion of the pituitary during the entire life of an individual, in largest quantities during puberty. It is synthesized as a precursor, and released into the blood following modifications. Human growth hormone is used in the treatment of certain forms of dwarfism caused by its deficiencies, in obesity therapy and in wound and burn treatment. Due to the fact that only human growth hormone can be used in the treatment of growth abnormalities, a large demand exists for this compound.
Until recently, the only source of hGH were cadaver pituitaries, whose hormone was isolated using time consuming and complicated technologies. The efficiency of producing hGH from pituitaries is very low, since only 2-3 mg of hormone are obtained from one gland. recently, a series of methods have been designed to by pass this system. In U.S. Pat. No. 4,124,448, a method was described of producing hGH on a large scale through culturing human pituitary cells in a liquid culture. In the patent described, a 20-fold efficiency is obtained per pituitary, when compared to the extraction from autopsy glands. A significant reason to search for other methods of obtaining human growth hormone is that the administration of somatotropin from human pituitaries (the material came from, among others, Africa) bears a risk of infection with various incurable diseases such as Creutzfeldt-Jakob disease or AIDS.
Methods exist for producing hGH through fermentation using organisms transformed using DNA recombination.
Improved recombinant production of hGH and methods of purification thereof are detailed in U.S. Pat. Nos. 4,534,258; 4,898,830; 5,424,199; and 5,795,745.
A variety of bacterial hGH expression methods using conventional fermentation and induction conditions have been proposed in the art.
Attempts to bacterially express human and bovine somatotropin in non-secretion systems using structural genes having the sequences of their cDNAs were successful after introducing silent mutations into the front end of the structural gene attributed the original difficulties to translation being impeded by the secondary structure of the mRNA corresponding to the cDNA, and taught lessening such secondary structure to enable significant expression. DNA, Vol. 2, No. 1, 1983, pp. 37-45; U.S. Pat. Nos. 5,254,463 and 5,260,201.
EP1400593 describes a method of producing human growth hormone or a salt thereof, which comprises culturing the transformant Escherichia coli mM294/pNP3GHNO12 (FERM BP-7611). A promoter of the transformant has a novel nucleotide sequence between any −35 region sequence and any −10 region sequence. The promoter has a strong promoter activity and thus a target peptide or protein can be produced at a high efficiency and in large amounts through linking the structural gene encoding the target peptide or protein downstream of the promoter. Another mode of use of E. coli host cell for expression of the gene for somatotropin protein was proposed in EP0418219B1. The invention involves to E. coli host cell comprising a mutation of the rpoH or hf1B gene and a plasmid consisting of a mutant R1 replicon from pBEU-17 or pBEU-50, an origin of replication from pBR322, and cDNA encoding the heterologous protein. WO9726334A1 provides methods for the production of heterologous polypeptides including human growth hormone, using a variety of recombinantly engineered mammalian secretory cell lines and blocking expression of native sequence and replacing with a construct giving improved secretion, useful to treat, e.g. diabetes.
An alternative approach for somatotropin production involves obtaining inclusion bodies. US20030229210A1 discloses a process for the preparation of active somatotropin from inclusion bodies of a recombinant host cell containing an inactive form of said somatotropin protein. The process comprises: contacting the inclusion bodies with an aqueous alcohol solution to solubilize said protein and bringing the solubilized protein into contact with a mild oxidizing agent to refold and form intramolecular disulfide bonds between cysteine residues of said protein.
Cleaval trials have been made to provide increased conformational and chemical stability of somatotropin. WO 94/10200 suggests a number of amino acid substitutions in somatotropin. Alterations of these characteristics are obtained via modification of somatotropins by site-directed mutagenesis.
As to purification of the growth hormone, methods for purifying and recovering biologically active somatotropin monomers from refold solution involve the solubilization and naturation of refractile bodies of host cells produced by recombinant DNA methodology. The purification process is based on the discovery that somatotropin monomers and somatotropin oligomers having overlapping isoelectric points may nevertheless be separated by selective precipitation over a narrow pH range (U.S. Pat. Nos. 5,182,369, 5,773,588).
Despite significant progress made in this area over the last few years, production of recombinant human growth hormone through expression in cellular expression systems is still in need of perfection. The main problems in need of resolution are to increase the overall efficiency of the whole process, as well as designing a method of increasing the stability of the protein produced. Growth hormone is a protein sensitive to factors which occur during its production and purification, which makes it difficult to produce and causes great losses in the amounts of active protein produced.
In biotechnology, the most useful vectors are the so-called expression vectors, which facilitate efficient synthesis of proteins encoded by the genes contained on the vector. Such vectors bear promoter sequences which facilitate transcription and translation, and sequences ensuring the stability of the synthesized protein. There are expression vectors under the control of strong promoters, whose synthesis can lead to accumulations of a given protein totaling 30% or even more of total cellular protein. Such vectors have been used for years in the production of many well known and useful proteins, particularly ones with desirable pharmacological properties. It is particularly desirable to provide new plasmids which could be used to produce new constructs useful in microbiological production of growth hormone, especially ones facilitating stable or regulated expression thereof. In this context it is particularly desirable to provide autonomic functional elements which could be used in the production of other useful constructs. For example, it is still desirable to produce transcription regulatory elements, like strong transcriptional promoters.
Protease UBP1 is an enzyme isolated from yeast, which cleaves ubiquitin from proteins fused to its C-end. The enzyme was described in 1991 (J. Tobias, A. Varshavsky, J. Biol. Chem. 1991, 266; 12021-12028) and is the subject of the patent application WO91/17245 (European patent EP 531 404). Its activity and culture conditions were described in E. coli. In accordance to the contents of the description, it is a cysteine protease, which binds ubiquitin with an ester bond during the course of the reaction. UBP1 is 809 amino-acids long. The enzyme's activity is dependent on its ability to cleave the ubiquitin peptide from a polypeptide fused to its C-end, regardless of the amino-acid sequence of the N-end of the polypeptide being digested off.
Application No. WO93/09235 describes other yeast proteins belonging to the same family of proteases, namely UBP2 and UBP3. These proteins exhibit similar activity (see also U.S. Pat. Nos. 5,494,818, 5,212,058, 5,683,904).
There are expression systems known, in which fusion proteins are obtained composed of ubiquitin or its derivative and a polypeptide of interest, and then, using a ubiquitin-removing enzyme (eg. UBP1) the protein of interest is recovered (for examples see: U.S. Pat. Nos. 5,132,213, 6,018,102). This method has many advantages such as improved quality and efficiency of obtaining the protein, as well as simplification of purification, which is of great importance in the industrial production of recombinant proteins (for example see: WO03/010204). Using an enzyme which removes ubiquitin and appropriate fusion proteins one may also obtain N-terminally modified polypeptides (for example: U.S. Pat. No. 5,847,097).
The international submission published as WO 2004/097011 describes UBP1 protease deletion mutants, containing a deletion of at least a portion of the initial 54 amino-acids from the amino-acid sequence of the UBP1 protease, and also describes some point mutations, which improve the expression level of such a protease in microbiological expression systems.
The application of an enzyme which removes ubiquitin in technological processes requires large amounts of this protein, which should also exhibit the maximal proteolytic activity level. A majority of known methods do not facilitate the efficient expression of this enzyme, which has greatly limited its applicability, especially in industrial processes. The purity and activity of the enzyme are important, if it is to be used in a subsequent stage of the production of a particular protein, particularly human growth hormone protein. Despite the solutions presented in WO 2004/097011, there is still a need to obtain a protein for cleaving UBP1, which could be produced in an efficient manner, for example through the expression in known microbiological systems, which protein would also exhibit improved specific proteolytic activity characteristics.
To summarise, it should be stated that the goal of the present invention is to propose an efficient method of obtaining human growth hormone human growth hormone by way of expression in bacterial cells, particularly in E. coli. Such a method should facilitate the production of relatively large quantities of protein of appropriate quality and activity for its application in the production of pharmaceutical preparations.
In particular, it is desirable to produce tools necessary to carry out such a method, especially efficient expression promoters, vectors containing them, as well as other tools such as enzymes, which are essential to the production of growth hormone through expression in a bacterial cell, encompassing the production of a large amount of stable fusion protein.
Therefore, a particular goal of the present invention is to also deliver a new, improved ubiquitin-cleaving enzyme cleaving ubiquitin, which would also be characterised by improved specific proteolytic activity, and which could be successfully used in the industrial production of growth hormone expressed in bacterial cells as fusion proteins with ubiquitin.
The next goal of the present invention is to propose the subsequent stages of the production of growth hormone, particularly renaturation conditions and purification of the protein produced.